Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

2

National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.

3

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland Baltimore, Baltimore, Maryland, USA.

4

Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.

5

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland Baltimore, Baltimore, Maryland, USA.

6

Department of Microbial Pathogenesis, School of Dentistry, University of Maryland Baltimore, Baltimore, Maryland, USA rkernst@umaryland.edu yod4@pitt.edu.

7

Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA rkernst@umaryland.edu yod4@pitt.edu.

Abstract

mcr-1 was initially reported as the first plasmid-mediated colistin resistance gene in clinical isolates of Escherichia coli and Klebsiella pneumoniae in China and has subsequently been identified worldwide in various species of the family Enterobacteriaceaemcr-1 encodes a phosphoethanolamine transferase, and its expression has been shown to generate phosphoethanolamine-modified bis-phosphorylated hexa-acylated lipid A in E. coli Here, we investigated the effects of mcr-1 on colistin susceptibility and on lipopolysaccharide structures in laboratory and clinical strains of the Gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens, which are often treated clinically by colistin. The effects of mcr-1 on colistin resistance were determined using MIC assays of laboratory and clinical strains of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa Lipid A structural changes resulting from MCR-1 were analyzed by mass spectrometry. The introduction of mcr-1 led to colistin resistance in E. coli, K. pneumoniae, and A. baumannii but only moderately reduced susceptibility in P. aeruginosa Phosphoethanolamine modification of lipid A was observed consistently for all four species. These findings highlight the risk of colistin resistance as a consequence of mcr-1 expression among ESKAPE pathogens, especially in K. pneumoniae and A. baumannii Furthermore, the observation that lipid A structures were modified despite only modest increases in colistin MICs in some instances suggests more sophisticated surveillance methods may need to be developed to track the dissemination of mcr-1 or plasmid-mediated phosphoethanolamine transferases in general.

MS analysis of WT E. coli strain YD626 (A) and the strain expressing mcr-1 (B). PEtN-modified species are represented in red (m/z 1,919 and 1,947). Proposed structures of WT E. coli lipid A (C) and PEtN-modified lipid A (D). Location of PEtN is suggestive; blue represents the lost phosphate moiety, the position of which is also suggestive. Asterisk (*) represents lipid A structure that varies in acyl chain length or hydroxylation status.